Oral vaccine comprising antigen surface-associated with red blood cells

ABSTRACT

Red blood cells or derivatives thereof such as ghost preparations can act as a potent carrier for orally administered antigens. Mucosal immunity in particular can be effectively induced against such viruses as influenza when adsorbed to chicken red blood cells and orally administered.

This invention relates generally to vaccines and in particular, althoughnot limited to, influenza vaccines appropriate for oral administration.

Currently, most vaccines are administered parenterally with consequentproblems arising from the invasive nature of the administration route.For instance, it has been proposed, in U.S. Pat. No. 4,157,390 to usered blood cells as vehicles for the presentation of enteropathogenicantigens in a parenteral vaccine administered to prepartum sows. Afurther disadvantage of parenteral administration is that, in general,it induces a better blood borne immune response than a mucosal one, yetthe prophylaxis or treatment of some infective agents is moreappropriately dealt with by a strong mucosal immunity. The parenteralnature of prior art vaccines has not in general resulted in a strongmucosal response.

To redress some of these problems, orally administered vaccines havebeen proposed against various infective agents. Unfortunately as thealimentary tract provides a hostile environment, it is onlyfortuitously, such as in the Sabin vaccine, that these have beeneffective.

It is an object of the present invention to provide a vaccine whichredresses some of the disadvantages experienced in the past.

It has now been found that red blood cells and their derivatives canprovide a potent orally administered vehicle for the presentation ofantigens to the mucosal immune system. Therefore in accordance with afirst aspect of the invention there is provided an oral vaccinecomprising antigen surface-associated with red blood cells orderivatives thereof.

A second aspect of the invention provides a method of eliciting animmunoresponse, particularly a mucosal immune response, in a mammal themethod comprising the oral administration of a vaccine as definedimmediately above.

In general, the antigen will derive from an infectious agent. Such aninfectious agent may be a virus, in particular those viruses in which amucosal immunoresponse appears important in prophylaxis or acuteinfection. Examples of these include respiratory viruses such asinfluenza or rhinovirus, polio and certain gastrointestinal infectionssuch as rotavirus. Other infections of mucosal like tissue include E.coli infections of the urinary tract and chlamydia infection of the eyein trachoma. The present invention can also invoke a systemic immunitysuggesting uses in systemic infections such as hepatitis or tetanus.Other immunizing applications such as those intended for anti-allergy orcontraceptive treatment may also be appropriate. The antigen maycomprise a plurality of antigens to produce a multivalent vaccine forall strains of, for instance, influenza, present in a season.Alternatively or additionally, the antigen may comprise a plurality ofantigens from different organisms thus leading to a single vaccineeffective against more than one disease or condition.

The invention is not limited to the use of whole red blood cells in thevaccine, because derivatives thereof, such as ghosts or membranepreparations, can also yield the desired enhanced mucosalimmunogenicity. One observation has been that the use of red blood cellsprovides extremely uniform size particles within the optimal 5 to 10 μmrange optimally taken up by Peyers patches, see, for instance Jones etal. (1988) Scand. J. Immunol., 27, 645. Thus, by the use of theinvention, an antigen can be effectively targeted to the Peyers patches,the "mucosal motor" for the activation of the common mucosal system.Previous attempts to target Peyers patches have failed due todifficulties in creating discrete uniform size particles, as shown byKreuter et al. (1981) J. Pharmaceutical Sci., 70, 367.

An advantage of the orally administered red blood cell (or derivative)system of the present invention is that due to previous dietaryexposure, the red blood cells are immunologically well tolerated by mostindividuals. It is therefore desirable that the red blood cells shouldoriginate from farm animals such as chickens, ducks, cows or sheep.Alternatively, to ensure hypoallergicity human red blood cells could beused.

The surface-association of the antigen with the red blood cell orderivative may take place through adsorption or binding via liganding orother chemical modification. One alternative is to bind the antigen to alectin or antibody (fragment) having specificity for red blood cellmarkers. Preferably, however, the association takes place through theinteraction of an indigenous (i.e. naturally present) receptor on thered blood cell, the receptor having specificity either for the antigenitself or for a linking or haptenic group attached to the antigen.

For instance, the influenza haemagglutinin glycoprotein (HA) bindsavidly to a surface receptor of chicken red blood cells (CRBC).Influenza preparations, either live attenuated or inactivated, can bedirectly bound via the HA and surface receptor to the red blood cell (orderivative). Virus purification from culture supernates and vaccinepreparation can be achieved in a single step by the simple addition ofthe red blood cell (or derivative) to the supernate. A preferredtechnique for preparing viral antigens utilizes gamma irradiation whichappears to favourably maintain the antigenicity of the preparation.

The efficacy of antigen presentation on the red blood cell (orderivative) is such that very small amounts of antigen may induce aneffective response, suggesting a potentiation or adjuvancy of thissystem. The small amounts of antigen required may allow the provision ofthe multivalent vaccines discussed above.

An unexpected feature of the present invention is that it has been shownto provide effective local and systemic stimulation, resulting incirculating and local antibodies which have a demonstrated prophylacticeffect.

An embodiment of a vaccine in accordance with the invention will now bedescribed by way of example only with reference to the following exampleand the accompanying FIGS. 1 to 11 which illustrate the results of thevarious experiments discussed in the Example. In particular

FIG. 1 is a bar graph depicting the protection of mice to H3N2 virusesafter oral vaccination with CRBC adsorbed with A/Qld/6/72 virus;

FIG. 2 is a bar graph depicting virus specific antibody titres of lunghomogenates from mice after oral vaccination with virus adsorbed CRBC;

FIGS. 3A and 3B are bar graphs depicting lung antibody titre andprotection of mice following oral vaccination with graded doses of CRBCadsorbed with A/Qld/6/72 virus;

FIG. 4A and 4B are bar graphs depicting a similar experiment to FIG. 3except the mice were immunised subcutaneously;

FIG. 5 is a bar graph depicting the protection of mice to A/Qld/6/72,A/Vic/36/88 and A/HK/123/77 viruses following oral immunisation withtriple adsorbed virus;

FIG. 6 is a bar graph depicting virus antibody specific for A/Qld/6/77,A/Vic/36/88 and A/HK/123/77 viruses following oral immunisation withtriple adsorbed virus;

FIG. 7 is a bar graph of a comparative experiment depicting crossprotection of mice to H2N2 and H1N1 viruses after oral vaccination withlive H3N3 viruses (A/Qld/6/72);

FIG. 8 is a bar graph depicting cross protection against heterotypicchallenge in mice 3, 6 and 12 weeks after oral administration withA/Qld/6/72 (H3N3);

FIG. 9 is a bar graph depicting haemagglutination inhibition titre inlung homogenates of mice following oral immunisation with CRBC adsorbedwith A/Qld/6/72 (H3N3) virus;

FIG. 10 is a bar graph depicting homotypic cross protection induced byA/Qld/6/72 (H3N2) to differing viruses within the H3N2 subtype; and

FIG. 11 is a bar graph depicting correlation between antibody titre andhomotypic cross protection over time.

EXAMPLE

ORAL IMMUNISATION WITH INACTIVATED INFLUENZA VIRUSES USING CHICKEN REDCELL PARTICLES AS CARRIERS

This Example is based on the ability of influenza virus, irrespective ofantigenic drift, to bind to chicken red blood cell surface receptorthrough the haemagglutinin glycoprotein. Thus virus purification andvaccine preparation can be achieved in one single step. Briefly, chickenred blood cells were washed three times with phosphate-buffered saline(PBS) and hen resuspended to 10¹⁰ cells/ml in PBS. They were then usedwhole for virus adsorption or as `ghosts` after lysis with Tris-bufferedammonium chloride solution. Adsorption was carried out at roomtemperature for 30 minutes with various dilutions of gamma irradiated(2×10⁶ rads, ⁶⁰ Co) allantoic fluid containing virus grown inspecific-pathogen free eggs. After three washes with PBS to remove eggmaterial and excess virus, virus-adsorbed CRBC were then resuspended in2% sodium bicarbonate solution to 5×10⁸ particles/ml.

For oral vaccination, Swiss male mice were administered 0.4 mlphosphate-buffered (unimmunised control) or 0.4 ml inactivated virusalone or CRBC (whole or lysed) absorbed with gamma irradiated virus(immunised) in sodium bicarbonate on each of days 1, 3 and 5. Ten daysafter the final dose the mice were challenged with live virusintranasally by inoculating 50 μl of live virus suspension inphosphate-buffered saline into each nostril using a micropipette. Fourdays after challenge, mice were killed. Virus and antibody titres in thelung homogenates were determined by MCDK virus infectivity assay andELISA.

A representative experiment is shown in FIGS. 1 and 2. Mice immunizedwith PBS or inactivated virus were not protected against challenge withlive virus in the respiratory tract compared to mice immunized withCRBC-adsorbed with gamma irradiated viruses. Further, there was acorrelation between protection and the presence of virus-specificantibodies in respiratory secretion (FIG. 2). Protection and antibodyresponse were dose-dependent, with mice being protected when immunizedwith CRBC adsorbed with virus as low as log₁₀ 2 TCID as depicted inTable 1 which shows the antibody response and protection of mice fromlung infection following oral vaccination with graded doses ofA/Qld/6/72 virus adsorbed to CRBC.

                                      TABLE 1                                     __________________________________________________________________________    Antibody response and protection of mice from                                 lung infection following oral immunization with CRC                           absorbed with graded doses of inactivated A/Qld/6/72                          (H3N2) virus                                                                  A/Qld/6/72 (H3N2)                                                             Immunization                                                                  Dose      Lung         Nasal Wash                                             (Log.sub.10 TCID.sub.50)                                                                IgG (EU/ml)                                                                          PFU/Lung                                                                            IgA (EU/ml)                                                                          PFU/ml                                          __________________________________________________________________________    0          4.8 ± 0.002                                                                      6.06 ± 0.08                                                                       5.6 ± 0.09                                                                       4.6 ± 0.15                                   <1        17.2 ± 0.79                                                                       5.40 ± 0.24                                                                      10.1 ± 0.59                                                                       4.4 ± 0.23                                   2         67.5 ± 11.3                                                                       2.67 ± 0.84                                                                      51.6 ± 0.13                                                                       <2                                              3         65.9 ± 8.5                                                                        <2    42.9 ± 14.3                                                                       <2                                              4         65.4 ± 8.4                                                                        <2    14.9 ± 0.50                                                                       <2                                              __________________________________________________________________________

By comparing FIGS. 3 and 4 it can be seen that lung antibody titrefollowing oral vaccination with the product of this invention isenhanced over that seen from subcutaneous administration. Theversatility of the CRBC carrier system was demonstrated in an experimentin which triple absorbed virus CRBC was tested using A/Qld/6/72,A/Vic/36/88, and A/HK/123/77 viruses. All three viruses were clearedfrom the murine lung following challenge and the responses correlatedwith virus-specific antibody levels in respiratory secretion (FIGS. 5and 6 ). Clearly, virus-absorbed CRBC particles are potent inducers ofprotective immunity in the respiratory tract when administered by theoral route. Table 2 on page 9 includes preliminary data from currentstudies, indicating cross protection following oral immunisation withabsorbed virus.

                                      TABLE 2                                     __________________________________________________________________________    Cross specific virus antibody* in respiratory secretion of mice               following                                                                     oral immunisation with A/6/Qld/2/72 (H3N2) influenza virus absorbed to        CRBC                                                                                                          *V(GT)007                                                                             *V(GT)-035/79                                   A/Vic/36/88                                                                          A/HK/123/77                                                                           A/Qld/2/72                                                                           (H6N1)  (H2N2)                                          (H1N1) (H1N1)  (H3N2) (Recombinant)                                                                         (Wild Variant)                                                                        *B/Vic/2/87                   __________________________________________________________________________    Unimmunised                                                                             --     --       4.6 ± 0.008                                                                      --      --                                    Immunised with                                                                          68.4 ± 8.5                                                                        78.5 ± 9                                                                           123 ± 20                                                                          86.3 ± 9.0                                                                         95.4 ± 9.5                                                                         7.6 ± 0.7                  A/Qld/6/72 (H3N2)                                                             absorbed CRBC                                                                 __________________________________________________________________________     *ELISA assay including whole virus                                       

Heterotypic cross protection and the duration of protection wereassessed by immunizing groups of mice with 4×10⁷ A/Qld/6/72 (H3N2) virusabsorbed CRBC and challenging each group with Log₁₀ 5.6 TCID A/Qld/6/72(H3N2), A/AA/6/60ca (H1N1) and A/HK/123/77 (H2N2) respectively. Threeweeks, 6 weeks and 12 weeks after oral immunisation, mice werechallenged with live wild-type viruses from different sub types. Asshown in FIG. 8 viruses were completely cleared from the lung. Virusclearance correlated with antibody against the homologous strain as wellas cross protective antibody against the heterologous strains. Similarresults were observed in the nasal wash in terms of virus clearance andantibody levels. The results were validated using the HI test todetermine antibody levels in lung homogenates and nasal washes, whichcorrelate with protection.

Homotypic cross protection induced by A/Qld/6/72 H3N2 to differingviruses within the H3N2 subtype is depicted in FIG. 10 while FIG. 11correlates antibody titre and homotypic cross protection in this system.

A remarkable level of cross protection (across the haemagglutininbarrier) was demonstrated using ELISA antibody assay. This crossprotection could be directly demonstrated using in vivo challenge. Thisprotection (antibody production and pulmonary clearance) could bedemonstrated to remain relatively undiminished for at least threemonths: following a primary immunization. Antigen specificity wasfurther examined using haemagglutination inhibition titres, and again,cross protection was demonstrated, indication an extraordinaryproduction of haemagglutinin specific antibodies (FIG. 9), although by12 weeks antibody titres were falling.

Thus it would appear that immunization with this embodiment of theinvention stimulates a broad immunity that crosses the "haemagglutininbarrier" of systemic immunisation using a very small Mount of virus. Byway of example, FIG. 7 indicates that cross protection is not seen inorally administered influenza vaccine in the absence of the red bloodcells (or derivatives).

Although the invention has been illustrated using a murine modelemploying gamma irradiated influenza virus in association with chickenred blood cell derivatives it will be appreciated that other systemsincluding human fall within the spirit and scope of the invention.

What is claimed is:
 1. An oral vaccine comprising an influenza antigen,wherein said influenza antigen is surface-associated with a red bloodcell or a ghost preparation thereof, and wherein said vaccine elicits amucosal immune response in mammals.
 2. A vaccine according to claim 1wherein the influenza antigen comprises influenza virus inactivated bygamma irradiation.
 3. A vaccine according to claim 1 wherein the antigenis adsorbed to the surface of the red blood cell or ghost preparationthereof.
 4. A vaccine according to claim 1 wherein the antigen ischemically coupled to the red blood cell or ghost preparation thereof.5. A vaccine according to claim 1 wherein the antigen binds anindigenous surface receptor of the red blood cell or ghost preparationthereof.
 6. A method for eliciting a mucosal immune response in amammal, said method comprising orally administering to said mammal avaccine as claimed in claim 1 in an amount effective to elicit saidmucosal immune response.
 7. The vaccine according to claim 1 whereinsaid influenza antigen is influenza type A.
 8. The vaccine according toclaim 1 wherein the red blood cell is a chicken red blood cell or ghostpreparation thereof.
 9. The vaccine according to claim 1 wherein the redblood cell is a duck red blood cell or ghost preparation thereof. 10.The vaccine according to claim 1 wherein the red blood cell is a cow redblood cell or ghost preparation thereof.
 11. The vaccine according toclaim 1 wherein the red blood cell is a sheep red blood cell or ghostpreparation thereof.
 12. The vaccine according to claim 1 wherein thered blood cell is a human red blood cell or ghost preparation thereof.13. An oral vaccine comprising an influenza Type A antigen, wherein saidinfluenza antigen is surface-associated with a red blood cell or a ghostpreparation thereof, and wherein said vaccine elicits a mucosal immuneresponse in mammals.